A Chimeric Antigen Receptor That Binds to a Conserved Site on MICA.

The NKG2D ligand MHC class I chain-related protein A (MICA) is expressed on many varieties of malignant cells but is absent from most normal tissues, and thus represents a potential target for chimeric Ag receptor (CAR) T cell-based therapeutics. However, there are more than 100 alleles of MICA, so the ability to target a conserved site is needed for a therapy to be used in most patients. In this study, we describe a fully human anti-MICA CAR created by fusing the single-chain fragment variable B2 to the full length DAP10 protein and the traditional CD3ζ signaling domain. Human T cells expressing the B2 CAR killed MICA-positive tumor cells, produced IFN-γ when in contact with MICA-positive tumor cells or plate-bound MICA protein, and inhibited PANC-1 growth in a mouse xenograft model. To localize B2's epitope on MICA, we used novel computational methods to model potential binding modes and to design mutational variants of MICA testing these hypotheses. Flow cytometry using a commercial anti-MICA/MICB Ab indicated that the variant proteins were expressed at high levels on transduced P815 cell lines. One variant protein (R38S/K40T/K57E) showed reduced staining with a B2-IgG1 fusion protein compared with controls and did not induce IFN-γ production by human T cells expressing the B2 CAR. These results show antitumor activity of MICA-specific CAR T cells and indicate an essential role for a conserved site in the exposed loop involving aa 38-57 of MICA. This study describes a novel MICA-specific CAR and discusses its potential use as a cancer therapeutic.

T-bet promotes potent antitumor activity of CD4+ CAR T cells.

Chimeric antigen receptor (CAR) therapy has shown promise against B cell malignancies in the clinic. However, limited success in patients with solid tumors has prompted the development of new CAR strategies. In this study, a B7H6-specific CAR was combined with different variants of T-bet, a transcription factor that acts as the master regulator to induce a Th1 phenotype in CD4+ T cells, to create more effective CAR T cells. Skewing CD4+ CAR T cells into a Th1 improved CAR T cell functional activity while promoting a robust proinflammatory response against B7H6-expressing tumors. The expression of T-bet with the B7H6-specific CAR in CD4+ T cells conferred higher expression of the CAR, elevated secretion of Th1 and proinflammatory cytokines, and improved cellular cytotoxicity against B7H6-expressing tumor cells. In vivo, CD4+ T cells co-expressing a B7H6-specific CAR and T-bet improved the survival of RMA-B7H6 lymphoma-bearing mice. Thus, CD4+ CAR T cells with increased T-bet expression have the potential to modify the tumor microenvironment and the immune response to better treat solid and hematologic cancers.

Chimeric antigen receptors with human scFvs preferentially induce T cell anti-tumor activity against tumors with high B7H6 expression.

B7H6 is emerging as a promising tumor antigen that is known to be expressed on a wide array of tumors and is reported to stimulate anti-tumor responses from the immune system. As such, B7H6 presents a good target for tumor-specific immunotherapies. B7H6-specific chimeric antigen receptors (CAR) based on a murine antibody showed successful targeting and elimination of tumors expressing B7H6. However, mouse single chain variable fragments (scFvs) have the potential to induce host anti-CAR responses that may limit efficacy, so human scFvs specific for B7H6 were selected by yeast surface display. In this study, we validate the functionality of these human scFvs when formatted into chimeric antigen receptors. The data indicate that T cells expressing these B7H6-specific human scFvs as CARs induced potent anti-tumor activity in vitro and in vivo against tumors expressing high amounts of B7H6. Importantly, these human scFv-based CARs are sensitive to changes in B7H6 expression which may potentially spare non-tumor cells that express B7H6 and provides the foundation for future clinical development.

Computationally-driven identification of antibody epitopes.

Understanding where antibodies recognize antigens can help define mechanisms of action and provide insights into progression of immune responses. We investigate the extent to which information about binding specificity implicitly encoded in amino acid sequence can be leveraged to identify antibody epitopes. In computationally-driven epitope localization, possible antibody-antigen binding modes are modeled, and targeted panels of antigen variants are designed to experimentally test these hypotheses. Prospective application of this approach to two antibodies enabled epitope localization using five or fewer variants per antibody, or alternatively, a six-variant panel for both simultaneously. Retrospective analysis of a variety of antibodies and antigens demonstrated an almost 90% success rate with an average of three antigen variants, further supporting the observation that the combination of computational modeling and protein design can reveal key determinants of antibody-antigen binding and enable efficient studies of collections of antibodies identified from polyclonal samples or engineered libraries.

Development of unique cytotoxic chimeric antigen receptors based on human scFv targeting B7H6.

As a stress-inducible natural killer (NK) cell ligand, B7H6 plays a role in innate tumor immunosurveillance and is a fairly tumor selective marker expressed on a variety of solid and hematologic cancer cells. Here, we describe the isolation and characterization of a new family of single chain fragment variable (scFv) molecules targeting the human B7H6 ligand. Through directed evolution of a yeast surface displayed non-immune human-derived scFv library, eight candidates comprising a single family of clones differing by up to four amino acid mutations and exhibiting nM avidities for soluble B7H6-Ig were isolated. A representative clone re-formatted as an scFv-CH1-Fc molecule demonstrated specific binding to both B7H6-Ig and native membrane-bound B7H6 on tumor cell lines with a binding avidity comparable to the previously characterized B7H6-targeting antibody, TZ47. Furthermore, these clones recognized an epitope distinct from that of TZ47 and the natural NK cell ligand NKp30, and demonstrated specific activity against B7H6-expressing tumor cells when expressed as a chimeric antigen receptor (CAR) in T cells.

How Chimeric Antigen Receptor Design Affects Adoptive T Cell Therapy.

Chimeric antigen receptor (CAR) T cells have been developed to treat tumors and have shown great success against B cell malignancies. Exploiting modular designs and swappable domains, CARs can target an array of cell surface antigens and, upon receptor-ligand interactions, direct signaling cascades, thereby driving T cell effector functions. CARs have been designed using receptors, ligands, or scFv binding domains. Different regions of a CAR have each been found to play a role in determining the overall efficacy of CAR T cells. Therefore, this review provides an overview of CAR construction and common designs. Each CAR region is discussed in the context of its importance to a CAR's function. Additionally, the review explores how various engineering strategies have been applied to CAR T cells in order to regulate CAR T cell function and activity. J. Cell. Physiol. 231: 2590-2598, 2016. © 2016 Wiley Periodicals, Inc.

B7H6-Specific Bispecific T Cell Engagers Lead to Tumor Elimination and Host Antitumor Immunity.

Substantial evidence showed that T cells are the key effectors in immune-mediated tumor eradication; however, most T cells do not exhibit antitumor specificity. In this study, a bispecific T cell engager (BiTE) approach was used to direct T cells to recognize B7H6(+) tumor cells. B7H6 is a specific ligand for the NK cell-activating receptor NKp30. B7H6 is expressed on various types of primary human tumors, including leukemia, lymphoma, and gastrointestinal stromal tumors, but it is not constitutively expressed on normal tissues. Data from this study showed that B7H6-specific BiTEs direct T cells to mediate cellular cytotoxicity and IFN-γ secretion upon coculturing with B7H6(+) tumors. Furthermore, B7H6-specific BiTE exhibited no self-reactivity to proinflammatory monocytes. In vivo, B7H6-specific BiTE greatly enhanced the survival benefit of RMA/B7H6 lymphoma-bearing mice through perforin and IFN-γ effector mechanisms. In addition, long-term survivor mice were protected against an RMA lymphoma tumor rechallenge. The B7H6-specific BiTE therapy also decreased tumor burden in murine melanoma and ovarian cancer models. In conclusion, B7H6-specific BiTE activates host T cells and has the potential to treat various B7H6(+) hematological and solid tumors.